As used in histology, a cryostat is a cooled chamber which is used to freeze a tissue specimen. The cryostat chamber is capable of maintaining a constant low temperature, typically by means of mechanical refrigeration. A specimen sample such as body tissue, bone, teeth, or sections of entire body organs are frequently desired to be frozen, then cut to a thin uniform thickness, to examine microscopically.
Examples of prior art cryostats are shown, for example, in U.S. Pat. Nos. 3,680,420; 4,840,035; 4,840,034; 3,495,490; and 3,462,969. Each of these prior art devices has shortcomings as will be generally discussed below.
In a typical prior art procedure for freezing, cutting and preparing a surgical specimen for microscopic examination, the tissue sample is brought into the laboratory for diagnosis while the patient is still on the operating table. Thus, as can be easily appreciated, it is essential to freeze, cut, and diagnose the section of tissue specimen as quickly as possible. Any unnecessary delays can be life threatening. In order to minimize the time required to perform the critical steps of freezing, cutting and diagnosis of a tissue section, an embedding medium (e.g., an aqueous solution, a viscous aqueous solution, or a viscous aqueous gel) is dispensed onto a specimen holder or block holder which is usually in the form of a small metal block. The specimen is either placed on top of or submerged into the embedding medium, and then is frozen by any number of means. In most prior art cryostats, the tissue specimen holder is placed on a cold shelf (the freezing shelf) where the medium and tissue specimen are frozen. Alternatively, a small heat extractor which has been equilibrated to the freezer shelf temperature, may be used to flatten and freeze the tissue specimen. Yet another technique may be used in which the entire specimen holder with the medium and tissue specimen in place, may be immersed in iso-pentane which is either chilled in a dry-ice bath or liquid nitrogen. In still another technique, direct immersion in liquid nitrogen may be used. As can be appreciated, regardless of the freezing technique incorporated, it is generally desirable that the tissue be frozen at the lowest possible temperature (and therefore as quickly as possible) since more rapid freezing results in the formation of smaller ice crystals, and therefore, less damage to the tissue morphology. Also, since the tissue itself is a poor thermal conductor, the best frozen tissue to examine is the tissue at the surface closest to the cooling source. The tissue layers which are further from the surface freeze more slowly (due to the low thermal conductivity of tissue) and therefore have larger ice crystals and poorer quality tissue morphology.
While certain cooling agents such as liquid nitrogen provide relatively rapid freezing, due to its boiling point of -196.degree. C., a thermal conductivity problem arises since the liquid will immediately become a gas upon contact with the tissue specimen. For this reason, direct metallic contact, when used as a means of freezing tissue, has been demonstrated as being superior to liquid immersion because of the higher thermal conductivity of metals compared to liquids. This direct metallic contact freezing technique is used in a snap-freezing device sold under the tradename "Gentle Jane" by Instrumedics, Inc. of Hackensack, New Jersey. The freezing plane of a chilled heat extractor is brought in contact with the specimen and surrounding gel and almost instantaneously freezes and flattens the sample.
During the freezing process a complex set of rapid events takes place during which time water is pulled out of the cellular material to form ice crystals. This transport of water dries out the cellular material (dehydration). As the ice-crystals form, their growth displaces the adjacent cellular material. In addition, the ice crystals, which have microscopic points and edges, can also puncture and/or cut the cell membranes. The slower the ice crystal formation (slow freezing) the more time there is for crystals to grow in size. As a result, there is considerable displacement of the original tissue architecture grossly and microscopically. In the conventional section retrieval method whereby the tissue section is melted, the melting ice crystals rehydrate the cells in a random manner thereby obliterating the original detail inherent in the cell. In those cells whose membranes have been damaged, there may be an additional outflow of fluids which causes collapse of the cell.
By contrast, very rapid or snap-freezing converts the identical amount of water into ice except the ice crystals form very rapidly and as a result are very small. Under ideal conditions in which the tissue is snap-frozen at 196.degree. C. and the uppermost section is retrieved, the ice crystals are so small that even under magnification of 1,000 times they are difficult to detect. Under these conditions, the ice crystal damage visually blends into the background of the cell structure.
The retrieval of a snap-frozen section at or very close to the freezing plane begins to approach the ideal. Heretofore it was not possible to do this. Typically, the section that is retrieved is deeper in the specimen where its freezing rate was much slower. Consequently the ice crystals are larger and the damage greater. The primary virtue of this freezing method in addition to its speed and level of automation is the ability to acquire a snap-frozen section at or close to the freezing plane.
After the tissue specimen has been frozen on the block holder, it is then manually clamped in a vice-like mechanism of a microtome apparatus. Through a series of synchronized physical movements, the microtome blade is used to cut the tissue specimen. The thickness of the individual sections attainable range typically between two microns and twenty-five microns.
In practice, before the actual cutting takes place, it is necessary to align the frozen surface of the tissue to the cutting plane defined by the knife edge. This step typically requires a cumbersome manual trial and error procedure requiring considerable patience and skill. The knife holder is temporarily unclamped and repositioned either closer to or further away from the frozen surface of tissue and then reclamped. The operator then makes a test cut to determine if further adjustment is necessary. The process is repeated as often as necessary until the frozen surface is positioned at the knife edge within acceptable limits.
During the normal trimming sequence, the uppermost layers of tissue are trimmed away in order to obtain a flat surface with a completely exposed tissue face. Even when the aforementioned direct metallic heat extractor technique is used, and a flat surface results, the freezing plane may not be coincident with the cutting plane of the microtome, thus requiring significant trimming before a desired flat specimen face is achieved. The need for additional trimming sequences not only results in the need to remove additional debris (which can be performed by the vacuum system described in U.S. Pat. No. 5,255,585) and additional time delays, but would also result in a poorer quality section for diagnosis since, as described previously, tissue sections further from the surface generally suffer greater morphological damage due to the slower freezing of these surface sections than those sections closer to the cold surface.
If there is a large angular misalignment between the freezing plane in the cryostat and the cutting plane of the microtome, the operator may have to realign the clamping of the block to bring the freezing plane into closer alignment with the cutting plane. After realignment of the block, the trimming sequence is repeated until a new smooth surface is attained. This block holder realignment and trimming of the block face can be the most time consuming step of the entire process thus potentially jeopardizing the well being of the patient.
Additional delays in the process often result due to the need to "tease" the cut section away from the block face and flatten it on the surface of the knife (i.e., the brush method). Alternatively, the "anti-roll-plate" method can be used in which a flat plate is positioned to create a gap into which the cut section can advance, thus reducing curling. Unfortunately, both methods are operator sensitive, requiring a skillful hand. Also, the thinner the section, the more fragile the step becomes, making flattening and handling even more difficult. Once an appropriate thin section of the specimen has been obtained, the next step is to mount it on a slide. This step is usually accomplished by touching the frozen section with a room-temperature microscope slide thereby instantaneously melting the section. Capillary forces adhere the section to the slide. The section on the slide is then usually dried, freezed, stained and cover-slipped to prepare it for microscopic examination.
It can be appreciated that the prior art does not provide a fast and effective technique for freezing, cutting, and examining a thin section of a tissue specimen for medical diagnostic purposes. All of the prior art devices require an undesirable amount of skilled manual handling of the tissue specimen which results in unreasonable delays. The problem is compounded by the poor morphological detail of relatively slow frozen tissue, making it difficult to give an unequivocal diagnosis.
It is therefore an object of the present invention to provide a new, faster, and improved system and method for freezing and cutting a tissue specimen for medical diagnostic purposes.
It is therefore another object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which performs both the snap-freezing and cutting steps in a single chamber.
It is yet another object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which minimizes manual handling of said tissue specimen.
It is a further object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which minimizes the amount of trimming required to be made by the operator to obtain an appropriate section for examination.
It is still another object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which freezes the tissue specimen in a manner which results in a minimal time delay and reduced ice crystal damage due to the smaller ice crystal size.
It is still a further object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which provides an appropriate tissue section with a minimal amount of trimming.
It is still another object of the present invention to provide a new and improved system and method of freezing and cutting a tissue specimen for medical diagnostic purposes which provides an appropriate tissue section with a large specimen face.
Further objects and advantages of the present invention will become apparent as the following description proceeds.